Our research objective is to obtain information concerning the mechanism and control of DNA replication in the slime mold Physarum polycephalum. We have been isolating temperature-sensitive (ts) mutant strains of this eukaryote for use in determining gene-function relationships with regard to DNA synthesis both in vivo and in vitro. Our specific research goals for this year are summarized below: 1. We will continue the routine screening of mutagenized outgrown amoebal clones for strains carrying ts mutations. We will also carry out reconstruction experiments to develop optimal conditions for replica plating and for suicide enrichment procedures. 2. The ts strains which we have obtained will be characterized by their growth kinetics at the restrictive and permissive temperatures. These amoebae will be used to construct homozygous ts diploid plasmodia which will be tested for expression of the ts phenotype. Those plasmodia which show temperature sensitivity with regard to growth will be tested for the effects of temperature on DNA replication and other macromolecular synthetic processes. 3. We will continue the characterization of cycloheximide-resistant and phleomycin-resistant amoebal strains as well as the characterization of plasmodia constructed from these amoebae.